Fragmentation patterns of selected ergot alkaloids by electrospray ionization tandem quadrupole mass spectrometry

Tall fescue toxicosis and other maladies in livestock result from the ingestion of vasoconstrictive ergot alkaloids produced by fungal endophytes associated symbiotically with the grass. In order to facilitate future analyses of grass extracts considered responsible for outbreak of related livestock diseases, we examined the electrospray ionization mass spectra of specific ergot alkaloids under conditions that permit protonation. Our purposes were both to record the spectra with interpretation of mechanisms of fragmentation and to derive commonalities that would allow the prediction of mass spectra of related compounds for which standards were not readily available. With [M + H](+) values in parentheses, water-insoluble lysergic acid peptide ergot derivatives ergovaline (m/z 534), ergotamine (m/z 582), ergocornine (m/z 562), ergocryptine (m/z 576) and ergocrystine (m/z 610) exhibited a consistent loss of water (-18 u) from the C-12' alpha-hydroxy functionality. Of this group, ergovaline and ergotamine generated an m/z 320 fragment deriving from cleavage of ring E amide and ether functions with retention of the peptide ring system methyl group. Ergocornine, ergocryptine and ergocrystine similarly formed an m/z 348 fragment with retention of isopropyl. These assignments were supported by the lack of similar fragments from the water-soluble ergot ergonovine, which lacks a peptide ring system. Clavine-type ergot alkaloids lysergic acid and lysergol lack any substituents beyond simple ones directly on the C-8 position and, similarly to ergonovine, lack significant fragments at m/z 268, 251 and 225 shared by the peptide ergot alkaloids.     

Electrospray[+] tandem quadrupole mass spectrometry in the elucidation of ergot alkaloids chromatographed by HPLC: screening of grass or forage samples for novel toxic compounds

Ergot alkaloids are mycotoxins generated by grass and grain pathogens such as Claviceps, for example. Ergot alkaloid-poisoning syndromes, such as tall fescue toxicosis from endophyte-infected tall fescue grass, are important veterinary problems for cattle, horses, sheep, pigs and chickens, with consequent impact on food, meat and dairy industries. Damage to livestock is of the order of a billion dollars a year in the United States alone. HPLC with UV and fluorescence detection are the predominant means of ergot alkaloid determination, with focus on quantitation of the marker compound ergovaline, although ELISA methods are undergoing investigation. These techniques are excellent for rapid detection, but of poor specificity in defining new or poorly characterized ergot alkaloids and related compounds. This paper demonstrates the facility of using electrospray(+) mass spectrometry with multiple reaction monitoring (MRM) detection during chromatographic examination of ergot alkaloid standards of lysergic acid, lysergol, ergonovine, ergovaline, ergotamine, ergocornine, ergocryptine and ergocrystine by HPLC. Ergoline-8 position epimers could be separated on the gradient HPLC system for ergocornine, ergocrystine and ergonovine and appeared as shoulders for ergotamine and ergovaline; epimers generally showed different patterns of relative intensity for specific MRM transitions. There was reasonable correspondence between retention of standards on the 2-mm ESI(+)MS phenyl-hexyl-based reverse phase column and those on the 4-mm C18-based column. Since up to 10% of clinical cases involving toxin exposure display unidentified chromatographic peaks, 11 samples of feed components associated with such cases were studied with developed MRM methods to attempt elucidation of crucial components if possible. Ergotamine appeared in all, ergovaline appeared in five and ergocornine appeared in six; ergonovine, ergocryptine, ergocrystine and lysergol also appeared in several. In addition, molecular weights of compounds newly revealed by mass spectrometry suggested ergosine, ergostine and ergoptine in four samples, for which standards were not available. Dehydrated products of ergotamine, ergocrystine and ergocornine were discovered, along with dihydrogenated ergocrystine and ergocryptine in seven of the samples, and the issue was raised as to whether dehydration was strictly an instrument-derived artifact. Finally, five of the samples, along with fescue seed standard, evidenced one or more of 14 new ergot alkaloids ranging in size from 381 to 611 molecular weight and with key mass spectral characteristics of ergot alkaloids, specifically the pair of peaks m/z 223 and 208, corresponding to the ergoline ring system and its demethylated variant, respectively. It is anticipated that findings such as these will provide impetus to future development of analytical methodology for these heretofore relatively rare ergot alkaloid species.     

Mass spectral characterization of ergot alkaloids by electrospray ionization, hydrogen/deuterium exchange, and multiple stage mass spectrometry: Usefulness of precursor ion scan experiments

Six ergot alkaloids belonging to the lysergic acid derivatives (ergonovine (EGN) and methysergide hydrogen maleinate (MHM)) and peptide-type derivatives (ergocristine (EGR), ergotamine (EGT), ergocornine (EGC) and alpha-ergokryptine (EGK)) were studied by positive electrospray tandem mass spectrometry. The fragmentation mechanisms of these compounds were studied by collision-induced dissociation (CID) using triple quadrupole and ion trap mass spectrometers, and the nature of the major product ions further confirmed by hydrogen/deuterium (H/D) exchange experiments. A common abundant product ion at m/z 223 was characteristic of the two classes of ergot alkaloids. Therefore, a precursor ion scan of m/z 223 that triggers information data acquisition (IDA) in combination with CID experiments was used to identify other potential ergot alkaloids. Using this approach, it was possible to confirm the presence of ergosine, another peptide-type ergot alkaloid, in a rye flour extract at trace levels.     

Synthetic Route to Ergot Alkaloids

Rye is sometimes infected by a fungus called Claviceps purpurea. The term ergot designates the dark, brown, tuberous bodies which can be collected before or during harvesting and represent one of the most remarkable drugs of our therapeutic arsenal. Actually, the most significant alkaloids are metabolic products of these fungi. We elaborated three alternative total synthetic pathways to construct the ergoline skeleton, one of which – suitable for scaling up – finally resulted in (+)‐lysergic acid ( 32a ) and α‐ergocryptine ( 1 ) (Schemes 5 and 6).     

Lysergic acids. II. Isolation and separation of lysergic acids (author's transl)]

During studies on the isolation of both lysergic acids, D-and D-iso-, from hydrolytic mixtures of ergot alkaloids, it became necessary to find a simple chromatographic system for column isolation of lysergic acids. The column with controlled pore glass is highly effective for these purposes. The separation of both isomeric lysergic acids occurs on the column with silica gel. The assay and the composition of lysergic acid are estimated by thin-layer chromatography on precoated plates.     

Ergot alkaloids: structure diversity, biosynthetic gene clusters and functional proof of biosynthetic genes

Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. They have been identi?ed in two orders of fungi and three families of higher plants. The most important producers are fungi of the genera Claviceps, Penicillium and Aspergillus (all belonging to the Ascomycota). Chemically, ergot alkaloids are characterised by the presence of a tetracyclic ergoline ring, and can be divided into three classes according to their structural features, i.e. amide- or peptide-like amide derivatives of D-lysergic acid and the clavine alkaloids. Signi?cant progress has been achieved on the molecular biological and biochemical investigations of ergot alkaloid biosynthesis in the last decade. By gene cloning and genome mining, gene clusters for ergot alkaloid biosynthesis have been identi?ed in at least 8 different ascomycete species. Functions of most structure genes have been assigned to reaction steps in the biosynthesis of ergot alkaloids by gene inactivation experiments or biochemical characterisation of the overproduced proteins.     

Radiolabelled peptide ergot alkaloids.

Paspalic acid ( 11 ), labelled at different positions with ³H or ¹⁴C and at specific activities up to 1 Ci/mmol (³H) and 2mCi/mmol (¹⁴C), has been prepared biosynthetically in a scale of 2–5 mmol in submerged cultures of a selected strain of Claviceps paspali by incorporation of DL -[5-³H]-tryptophan, DL -[6-³H]-tryptophan, DL -[alanine-2,3-³H]-tryptophan, or DL -[alanine-3-¹⁴C]-tryptophan. Radioactive lysergic acid ( 12 ) was obtained from paspalic acid by base catalysed rearrangement. The procedures for labelling the precursors at high specific activity arc described. The ergolene carboxylic acids 11 and 12 were used as key intermediates for tlie chemical synthesis of radiolabelled peptide ergot alkaloids 14 required for pharinacokinetic and metabolic studies. Linking of the aminocyclols 13 (peptide parts) of the ergotarnine (R¹ = methyl), ergoxine (R¹ = ethyl) and ergotoxine (R¹ =isopropyl) series with a reactive derivative of either lysergic acid or its mixture with paspalic acid was accomplished by standard procedures. l-Mctliyl-[13-³H]-ergotarnine (MY 25) ( 16 ) and 2-brotno-α-ergocryptine (bromocriptine, C73 154) ( 17 ), labelled with ³H at position 12 and with ¹⁴C in position 4, were obtained by alkylating ³H-ergolamine and by ljrominating appropriately labelled α-ergocryptines. Radioactive peptide ergot alltaloitls labelled by tlic present nietliotl proved suitable for the use in biological tracer studies.     

Layer-by-layer functionalized porous Zinc sulfide nanospheres-based solid-phase extraction combined with liquid chromatography time-of-flight/mass and gas chromatography-mass spectrometry for the specific enrichment and identification of alkaloids from Crinum asiaticum var. sinicum

The current widely utilized polymer or C8, C18 end-capped material-based sorbents for solid-phase extraction could not capture alkaloids well only based on “like dissolves like” principle. In this paper, a layer-by-layer functionalized porous Zinc sulfide nanospheres-based solid-phase extraction (SPE) combined with liquid chromatography time-of-flight/mass spectrometry (LC-TOF/MS) and gas chromatography-mass spectrometry (GC-MS) was developed for the specific enrichment and identification of alkaloids from complex matrixes, Crinum asiaticum var. sinicum crude extracts. The functionalized porous Zinc sulfide nanospheres were prepared by the amidation reaction of poly-(acrylic acid) (PAA) homopolymer with amino groups onto the porous ZnS nanospheres. Tandem LC-TOF/MS spectrometry presented that the almost all of the twenty-three main peaks in elution fraction from the SPE could be inferred as alkaloids with ion of mass according to the nitrogen rule and hit formula with Peak View1.2@software from AB SCIEX, and seven alkaloids including two new found chemical entities were directly identified from their GC-MS spectra and retention indices. We believe that this SPE protocol can also be utilized in the future to selectively enrich alkaloids from extracts of other plant species.     

Analysis of alkaloids in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiode array and tandem mass spectrometry detections

A new method based on accelerated solvent extraction (ASE) followed by ultra performance liquid chromatography (UPLC) analysis has been developed for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in sequential configuration. The operational parameters of ASE including extraction solvent, extraction temperature, static extraction time and extraction cycles were optimized. UPLC analysis was performed on an ACQUITY UPLC BEH C₁₈ column eluted by a mobile phase of acetonitrile spiked with a buffer solution consisting of 0.50% acetic acid and 20 mmol L⁻¹ ammonium acetate. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in C. chinensis Franch extracts. The samples were also analyzed on a high-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in C. chinensis Franch. Seven Huanglian samples from different locations were analyzed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of traditional Chinese medicine (TCM) herbs.     

Quantitative determination of five ergot alkaloids in rye flour by liquid chromatography-electrospray ionisation tandem mass spectrometry

A confirmatory method for detecting five ergot alkaloids, ergocristine, ergotamine, ergonovine, ergocornine and alpha-ergokryptine, in rye flour is described using high performance liquid chromatography coupled to tandem mass spectrometry detection by monitoring two transition reactions per analyte. The procedure entails a liquid-liquid extraction followed by a clean-up step using a C18 solid-phase extraction (SPE) cartridge. An analogue compound, methysergide hydrogen maleinate, was used to assess both repeatability sample preparation and potential MS response fluctuations. The method was fully validated according to the European Union (EU) criteria. Detection and quantification limits of all analytes were calculated ranging from 7 to 11 microg/kg and from 23 to 37 microg/kg, respectively. Fifteen rye flour samples were investigated with the newly developed method, and none of them were above the current Swiss limits of 200mg/kg for total ergot alkaloids.     

Rapid screening for cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for screening cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. at sub-ppm levels. An electrospray ionization orbitrap mass spectrometer, which provides accurate full scan MS and MS/MS data, was used in this study. After simple extraction with ethanol and purification by AB-8 resin, the extracts were subjected to LC/MS/MS analysis. A high mass tolerance (10 ppm) was used in the initial screening to filter the full scan MS data. The cyclo-dopa and diketopiperazine alkaloid standards gave limits of detection (LODs) at or below 5 ng/mL. The results also indicated that the method had an acceptable precision for day-to-day use in the identification of compounds. The alkaloids could be identified based on their MS/MS data, elemental compositions, and retention behavior. This system was used to assay trace amounts of cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L., leading to the identification of 5/2 confirmed/unconfirmed cyclo-dopa and 7/6 confirmed/unconfirmed diketopiperazine alkaloids, respectively. The screening method considerably reduces the time and cost involved in the identification of cyclo-dopa and diketopiperazine alkaloids in Portulaca oleracea L., as well as being a simple and convenient approach to the identification of other structural families of natural products.     

Characterization and identification of steroidal alkaloids in the Chinese herb Veratrum nigrum L. by high-performance liquid chromatography/electrospray ionization with multi-stage mass spectrometry

Electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) was performed to study the fragmentation behaviour of seventeen steroidal alkaloids (4 protoverine-type alkaloids, 10 germine-type alkaloids and 3 zygadenin-type alkaloids) from the Chinese herb Veratrum nigrum L. The MS(n) spectra of the [M+H](+) ions for steroidal alkaloids provided a wealth of structural information on the substituted groups. In positive ion mode, the three types of alkaloids showed very different characteristic ions: m/z 436 or 418 for protoverine-type alkaloids; m/z 438, 420 or 402 for germine-type alkaloids; m/z 440 or 422 for zygadenin-type alkaloids. These fragments were used to deduce their mass spectral fragmentation mechanisms. Furthermore, the primary compounds in methanolic extracts of the herb of Veratrum nigrum L. were investigated by using liquid chromatography (LC)/ESI-MS(n). As a result, 21 steroidal alkaloids (5 protoverine-type alkaloids, 14 germine-type alkaloids and 2 zygadenin-type alkaloids) were selectively identified from 27 determined peaks. Eleven compounds were unambiguously identified by comparing with standard compounds and ten compounds were tentatively identified or deduced according to their MS(n) data. Two of these compounds (xingangermine and deacetyl xinganveratrine) were found to be novel steroidal alkaloids. In addition, the chemical structures of two pairs of steroidal alkaloid isomers were deduced by comparing their fragment ions. Given the important structural information of known and unknown steroidal alkaloids in crude herbal extracts, this study is useful for identifying these types of steroidal alkaloids in crude materials rapidly and selectively.     

Synthetic strategies to the ergoline ring system of ergot alkaloids

The synthetic strategies to the ergoline ring system (I) have been outlined, and discussed in terms of their yields and limitations. The total syntheses of ergoline, racemic and natural dihydrolysergic acid, lysergic acid, isosetoclavine, penniclavine, elymoclavine and chanoclavine-I have been described. These illustrate the synthetic problems that have been encountered in ergoline chemistry. The role of key intermediates has been emphasised. Modification to the original schemes have not given yields or procedures that can compete with the production by fermentation of ergot alkaloids. However, it is hoped that the synthetic strategies discussed in this article will provide useful information, when considering new approaches, or modification, to the unique heterocyclic ring system of ergoline.     

Mechanism of alkaloid cyclopeptide synthesis in the ergot fungus Claviceps purpurea

Background: Previous analyses of the biosynthesis of the alkaloid cyclopeptides from the ergot fungus Claviceps purpurea were hampered by a lack of suitable systems for study in vitro, and this led to conflicting results concerning the mechanism of alkaloid cyclopeptide formation. Recently, D-lysergyl peptide synthetase (LPS) of the ergot fungus Claviceps purpurea, which assembles the non-cyclol precursors of the ergopeptines, has been partially purified and shown to consist of two polypeptide chains of 370 kDa (LPS 1) and 140 kDa (LPS 2); these contain all the sites necessary for the assembly of the D-lysergyl peptide backbone. The mechanism of D-lysergyl peptide synthesis remained unclear, however.Results: We have identified the obligatory peptidic intermediates in d-lysergyl peptide synthesis and the sequential order of their formation. The two LPS subunits catalyze the formation of d-lysergyl mono-, di-, and tripeptides as enzyme-thioester intermediates, the formation of which appears to be irreversible. Peptide synthesis starts when d-lysergic acid binds to the LPS 2 subunit, which most probably occurs after the previous round of synthesis has been completed by the release of the end product from the LPS enzyme.Conclusions: We have shown that the mechanism of d-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloid cyclopeptides.     

Identification of indole alkaloids in Nauclea officinalis using high-performance liquid chromatography coupled with ion trap and time-of-flight mass spectrometry

Indole alkaloids from Nauclea officinalis (Pierra ex Pitard) Merr are prospective agents for inflammation- related diseases. To speed up the process of discovering such bioactive compounds from natural origins, a mass spectrometry-based method was developed to screen N. officinalis for the presence of indole alkaloids. First, representative alkaloids were purified from herbs and analyzed using an ion trap (IT) mass spectrometer. Multi-stage mass spectra (MS(n))were utilized to establish the characteristic fragmentation pathways of indole alkaloids. It was shown that their fragmentation behaviors were correlated with the degree of unsaturation on ring-D of such alkaloids: if there is a double bond between C-3 and C-14, no fragments of ring cleavage are observed; otherwise, a fragment ion after reverse Diels-Alder cleavage of ring-D is observed as the base peak in their tandem mass spectrometry data. Then, herbal extracts of N. officinalis were analyzed using a combination of high- performance liquid chromatography (HPLC)/IT and HPLC/time-of-flight (ToF) systems. The structures of suspected alkaloids were deduced based on the accurate mass information from ToF and MS(n) from IT. Finally, a total of 10 indole and one indolizidine alkaloids were identified or tentatively characterized in extracts of N. officinalis. This work has demonstrated that the combination of MS(n) and ToF-MS can be used for rapid identification of monoterpenoid indole alkaloids in N. officinalis.     

Liquid chromatographic/mass spectrometric determination of biologically active alkaloids in extracts of Peschiera fuschiaefolia

Four alkaloids were isolated from an alcoholic extract of the bark from the stem of Peschiera fuschiaefolia. Two of these compounds, voacamine and voacamidine, are dimeric alkaloids which are thought to be responsible for the antimalarial activity of these extracts. The mass spectra and the response factors of these four compounds were obtained by liquid chromatography/mass spectrometry in the electrospray positive ionization mode. The concentrations of these alkaloids were measured in two different P. fuschiaefolia extracts. The ion chromatograms of the two extracts were compared on the basis of their [M + H](+) and [M + 2H](+2) ions characteristic of various alkaloids previously isolated from P. fuschiaefolia bark. The two extracts were found to differ mostly in the relative concentrations of the dimeric alkaloids.     

高效毛细管电泳-电喷雾飞行时间质谱联用分析黄连中的生物碱

建立了高效毛细管电泳-电喷雾飞行时间质谱联用(HPCE-ESI-TOF/MS)快速定性分析黄连中生物碱类化合物的分析方法. 使用未涂层石英毛细管, 以50 mmol/L乙酸铵-0.5%甲醇溶液(用氨水调至pH＝7.2)作为运行缓冲液, 分离电压为25 kV; 鞘液组成为50%甲醇-49.5%水-0.5%乙酸, 鞘液流速为4 μL/min; 质谱选用正离子模式, 碰撞电压(Fragmentor)为100 V. 结果表明, 通过各色谱峰紫外光谱和质谱测得精确分子量结果, 结合文献, 对黄连中7种生物碱进行了鉴定. 表明本方法简便、快速, 是黄连中生物碱类化合物快速分离、鉴别的有效方法.     

Characterization of the variation in the imidazole alkaloid profile of Pilocarpus microphyllus in different seasons and parts of the plant by electrospray ionization mass spectrometry fingerprinting and identification of novel alkaloids by tandem mass spectrometry

Pilocarpus microphyllus (Rutaceae), popularly known as jaborandi, is the only commercial source of an imidazole alkaloid named pilocarpine. In the present study, the variation in the profile of imidazole alkaloids in different seasons and in different parts of the P. microphyllus plant during the summer was analyzed by electrospray ionization mass spectrometry in the positive ion mode [ESI(+)-MS]. The fingerprints of these extracts repeatedly presented similar ions which were mass-selected and studied by tandem mass spectrometry (ESI-MS/MS and ESI-MS/MS/MS) and high-resolution mass spectrometry, resulting in the characterization of eight imidazole alkaloids. The data from the ESI(+)-MS fingerprints were analyzed by principal component analysis (PCA), showing that pilocarpine was present mainly in the summer, whereas in the autumn mainly pilosine and winter anhydropilosine were found. Three alkaloids, reported for the first time in extracts of P. microphyllus, were found. Analysis of the distribution of alkaloids in different parts of the plant during the summer showed that, although pilocarpine was present throughout the plant, 13-nor-8(11)-dihydropilocarpine was found mainly in the stem, pilosine and anhydropilosine were present mainly in the intermediary leaves, and the three new alkaloids were mainly found in the leaflets and petioles. Based on the dissociation patterns of these alkaloids, we observed that there were three structurally related groups of alkaloids differing in their distribution in the plant tissues and responding differently to seasonal variations. These results also indicate that these three groups of alkaloids could belong to intermediate, parallel or competitive pathways for pilocarpine formation biosynthesis.     

高效液相色谱-串联质谱法测定化妆品中10种生物碱

建立高效液相色谱-串联质谱同时测定化妆中10种生物碱的分析方法。实验优化了提取条件、净化方式和仪器条件等参数。样品经80%（v/v）甲醇水溶液超声提取，离心后过滤。采用Waters BEH C₁₈色谱柱分离，在多反应监测模式下测定，外标法定量。结果显示，在各自范围内，10种生物碱具有良好的线性关系，相关系数（R²）>0.9900，方法的检出限为5.0~12.5 μg/kg，定量限为12.5~50.0 μg/kg。在1倍、2倍和6倍定量限的加标水平下，10种生物碱的回收率为70.91%~116.75%，相对标准偏差为0.49%~9.98%（n=6）。该方法操作简单，适用于化妆品中10种有毒生物碱的快速筛查和定量分析。     

Liquid chromatography-particle beam electron ionization mass spectrometry method for analysis of botanical extracts: evaluation of ephedrine alkaloids in standard reference materials

The preliminary validation of a high-performance liquid chromatography particle beam mass spectrometry method (HPLC-PB/MS) with electron impact ionization source for analysis of botanical extracts is presented. The LC-PB/MS system was evaluated for the analysis of ephedrine alkaloids using ephedra-containing National Institute of Standards and Technology dietary supplement standard reference materials (SRMs) 3241 Ephedra Sinica Stapf Native Extract and 3242 Ephedra Sinica Stapf Commercial Extract. The ephedrine alkaloids were separated by reversed-phase chromatography using a phenyl column at room temperature. A linear gradient method with a mobile phase composition varying from 5:95 [MeOH:0.1% trifluoroacetic acid (TFA) in water] to 20:80 (MeOH:0.1% TFA in water) at a flow rate of 1.0 ml/min, with an analysis time of less than 20 min, was used. The source block temperature was evaluated to determine the optimal operating conditions by monitoring the intensities and fragmentation patterns of the ephedrine alkaloids. Ephedrine and N-methylephedrine were taken as a representative of the test alkaloids. The LODs on the sub-nanogram level were achieved, with ephedrine, pseudoephedrine, and methylephedrine in the SRMs quantified by a standard addition method with recoveries of > or = 86% and RSDs of < or = 14% (n = 3).